There is a rapidly growing interest in the detection of specific nucleic acid sequences. This interest has not only arisen from the recently disclosed draft nucleotide sequence of the human genome and the presence therein, as well as in the genomes of many other organisms, of an abundant amount of single nucleotide polymorphisms (SNP), but also from marker technologies such as AFLP and the general recognition of the relevance of the detection of specific nucleic acid sequences as an indication of for instance genetically inheritable diseases. The detection of the various alleles of the breast cancer gene BRCA 1 to screen for susceptibility for breast cancer is just one of numerous examples. The recognition that the presence of single nucleotide substitutions (and other types of genetic polymorphisms such as small insertion/deletions; indels) in genes provide a wide variety of information has also attributed to this increased interest. It is now generally recognised that these single nucleotide substitutions are one of the main causes of a significant number of monogenically and multigenically inherited diseases, for instance in humans, or are otherwise involved in the development of complex phenotypes such as performance traits in plants and livestock species. Thus, single nucleotide substitutions are in many cases also related to or at least indicative of important traits in humans, plants and animal species.
Analysis of these single nucleotide substitutions and indels will result in a wealth of valuable information, which will have widespread implications on medicine and agriculture in the widest possible terms. It is for instance generally envisaged that these developments will result in patient-specific medication. To analyse these genetic polymorphisms, there is a growing need for adequate, reliable and fast methods that enable the handling of large numbers of samples and large numbers of (predominantly) SNPs in a high throughput fashion, without significantly compromising the quality of the data obtained. One of the principal methods used for the analysis of the nucleic acids of a known sequence is based on annealing two probes to a target sequence and, when the probes are hybridised adjacently to the target sequence, ligating the probes.
The OLA-principle (Oligonucleotide Ligation Assay) has been described, amongst others, in U.S. Pat. No. 4,988,617 (Landegren et al.). This publication discloses a method for determining the nucleic acid sequence in a region of a known nucleic acid sequence having a known possible mutation. To detect the mutation, oligonucleotides are selected to anneal to immediately adjacent segments of the sequence to be determined. One of the selected oligonucleotide probes has an end region wherein one of the end region nucleotides is complementary to either the normal or to the mutated nucleotide at the corresponding position in the known nucleic acid sequence. A ligase is provided which covalently connects the two probes when they are correctly base paired and are located immediately adjacent to each other. The presence or absence of the linked probes is an indication of the presence of the known sequence and/or mutation.
Abbot et al. in WO 96/15271 developed a method for a multiplex ligation amplification procedure comprising the hybridisation and ligation of adjacent probes. These probes are provided with an additional length segment, the sequence of which, according to Abbot et al., is unimportant. The deliberate introduction of length differences intends to facilitate the discrimination on the basis of fragment length in gel-based techniques.
WO 97/45559 (Barany et al.) describes a method for the detection of nucleic acid sequence differences by using combinations of ligase detection reactions (LDR) and polymerase chain reactions (PCR). Disclosed are methods comprising annealing allele-specific probe pairs to a target sequence and subsequent ligation with a thermostable ligase. Amplification of the ligated products with fluorescently labelled primers results in a fluorescently labelled amplified product. Detection of the products is based on separation by size or electrophoretic mobility or on an addressable array.
More in particular, one of the disadvantages of the means and methods as disclosed by Barany et al. resides in the limited multiplex capacity when discrimination is based inter alia on the length of the allele specific probe pairs. Discrimination between sequences that are distinguishable by only a relatively small length difference is, in general, not straightforward and carefully optimised conditions may be required in order to come to the desired resolving power. Discrimination between sequences that have a larger length differentiation is, in general, easier to accomplish. This may provide for an increase in the number of sequences that can be analyzed in the same sample.
Other solutions that have been suggested in the art such as the use of circular (padlock) probes in combination with isothermal amplification such as rolling circle amplification (RCA) are regarded as profitable because of the improved hybridisation characteristics of circular probes and the isothermal character of RCA. The padlock probe is generally recognised as having superior characteristics compared to the conventional linear probes (Nilsson et al. Human mutation, 2002, 19, 410415; Science 1994, 265: 2085-2088).
However, providing for the necessary longer nucleotide probes for use as padlock probes is a further hurdle to be taken. In the art, synthetic nucleotide sequences are produced by conventional chemical step-by-step oligonucleotide synthesis with a yield of about 98.5% per added nucleotide. When longer probes are synthesised (longer than ca. 60 nucleotides) the yield generally drops and the reliability and purity of the synthetically produced sequence is generally recognised as a problem.
The specific problem of providing for longer probes has been solved by Schouten et al. (WO 01/61033). WO 01/61033 discloses the preparation of longer probes for use in ligation-amplification assays. They provided probes that are considerably longer than those that can be obtained by conventional chemical synthesis methods to avoid the problem associated with the length-based discrimination of amplified products using slab-gels or capillary electrophoresis, namely that only a small part of the detection window/resolving capacity of up to 1 kilo base length is used when OLA probes are synthesised by chemical means. With an upper limit in practice of around 100-150 bases for chemically synthesised oligonucleotides according to the current state of technology, this results in amplification products that are less than 300 base pairs long at most, but often much less (see Barany et al). The difficulty of generating such long probes (more than about 150 nucleotides) with sufficient purity and yield by chemical means has been countered by Schouten et al., using a method in which the probes have been obtained by an in vivo enzymatic template directed polymerisation, for instance by the action of a DNA polymerase in a suitable cell, such as an M13 phage. This is then followed by restriction enzyme digestion by providing a short oligonucleotide sequence to create a partially double stranded sequence to create a phosphorylated 5′ end of the long probe.
However, the production and purification of such ‘biological probes’ requires a collection of suitable host strains containing M13 phage conferring the desired length variations and the use of multiple short chemically synthesised oligonucleotides in the process, such that their use is very laborious and time-consuming, hence costly and not suitable for high-throughput assay development.
Another disadvantage of the use of circular probes is that the use of rolling circle amplification (RCA) which is commonly associated with padlock probes result in the formation of long concatamers. Examples thereof are inter alia U.S. Pat. No. 5,876,924, WO 98/04745 and WO 98/04746 by Zhang et al. who describe the ligation of circular or circularizable probes. Zhang et al. discloses the amplification of circular probes using oligonucleotide primers in RCA, using a DNA polymerase with strand displacement activity, thereby generating a long concatamer of the circular probe, starting from extension of the first primer. A second primer subsequently hybridises to the long concatamer and elongation thereof provides a second generation of concatamers and facilitates exponential amplification. Detection is generally based on the hybridisation of labelled probes. However, this method has proven to be less desirable in high throughput fashion. One of the reasons is that, for a high throughput method based on length discrimination, the use of RCA results in the formation of long concatamers. These concatamers are problematic, as they are not suitable for high throughput detection based on length based detection as this requires an additional preparation step (e.g. restriction enzyme digestion) in order to create a clearly detectable amplification product.
U.S. Pat. No. 6,221,603 disclosed a circular probe, which contains a restriction site. The probe is amplified using RCA and the resulting concatamers are restricted at the restriction site. The restriction fragments are then separated by length and detected. Separation and detection is performed on a capillary electrophoretic platform, such as the MegaBACE equipment available from Molecular Dynamics Amersham-Pharmacia. For detection (expensive) labelled dNTPs may be incorporated into the fragments during amplification, or the fragments may be detected by staining or by labelled detection probes. Digestion by the restriction enzyme is an additional step in the method for the successful detection of the target sequences and this extra step may affect the reliability of the method. Furthermore, the methods for labelling of the fragments as disclosed in U.S. Pat. No. 6,221,603 do not allow to fully utilise the capacity of simultaneous detection of multiple colours provided by most detection platforms such as the MegaBACE or others.
Accordingly, there is a need for oligonucleotide probes that combine the advantages of the various ligation probe types described herein. It is one of the goals of the present invention to provide such probes. It is another goal of the present invention to avoid the disadvantages of the commonly known probes as mentioned hereinbefore, in particular the unreliable or laborious chemical or enzymatic synthesis of relative long oligonucleotides. It is a further goal of the invention to provide for probes that are suitable for high throughput detection methods. It is also a goal of the present invention to provide for efficient, reliable and/or high throughput method for the detection of target nucleotide sequences, preferably by performing oligonucleotide ligation assays.
The present inventors have set out to eliminate or at least diminish the existing problems in the art while at the same time attempting to maintain the advantageous aspects thereof, and to further improve the technology. Other problems in the art and solutions provided thereto by the present invention will become clear throughout the description, the figures and the various embodiments and examples.